Methods for chromosome-specific staining

ABSTRACT

Methods and compositions for chromosome-specific staining are provided. Compositions comprise heterogenous mixtures of labeled nucleic acid fragments having substantially complementary base sequences to unique sequence regions of the chromosomal DNA for which their associated staining reagent is specific. Methods include methods for making the chromosome-specific staining compositions of the invention, and methods for applying the staining compositions to chromosomes.

The United States Government has rights in this invention pursuant toContract No. W-7405-ENG-48 between the U.S. Department of Energy and theUniversity of California, for the operation of Lawrence LivermoreNational Laboratory.

RELATED APPLICATION

This application is a continuation of U.S. Ser. No. 06/937,793, filedDec. 4, 1986, now abandoned, which, in turn, is a continuation in partof U.S. Ser. No. 819,314, filed Jan. 16, 1986, now abandoned The priorapplications were filed by the inventors named herein and were assignedto the same assignee. Priority is hereby claimed in said prior filedapplications.

BACKGROUND OF THE INVENTION

The invention relates generally to the field of cytogenetics, and moreparticularly, to methods for identifying and classifying chromosomes.

Chromosome abnormalities are associated with genetic disorders,degenerative diseases, and exposure to agents known to causedegenerative diseases, particularly cancer, German, "Studying HumanChromosomes Today," American Scientist, Vol. 58, pgs. 182-201 (1970);Yunis, "The Chromosomal Basis of Human Neoplasia," Science, Vol. 221,pgs. 227-236 (1983); and German, "Clinical Implication of ChromosomeBreakage," in Genetic Damage in Man Caused by Environmental Agents,Berg, Ed., pgs. 65-86 (Academic Press, New York, 1979). Chromosomalabnormalities can be of three general types: extra or missing individualchromosomes, extra or missing portions of a chromosome, or chromosomalrearrangements. The third category includes translocations (transfer ofa piece from one chromosome onto another chromosome), and inversions(reversal in polarity of a chromosomal segment).

Detectable chromosomal abnormalities occur with a frequency of one inevery 250 human births. Abnormalities that involve deletions oradditions of chromosomal material alter the gene balance of an organismand generally lead to fetal death or to serious mental physical defects.Down's syndrome is caused by having three copies of chromosome 21instead of the normal 2. This syndrome is an example of a conditioncaused by abnormal chromosome number, or aneuploidy. Chronicmyelogeneous leukemia is associated with the exchange of chromosomalmaterial between chromosome 9 and chromosome 22. The transfer ofchromosomal material in this leukemia is an example of a translocation.Clearly, a sensitive method for detecting chromosomal abnormalitieswould be a highly useful tool for genetic screening.

Measures of chromosome breakage and other aberrations caused by ionizingradiation or chemical mutagens are widely used as quantitativeindicators of genetic damage caused by such agents, BiochemicalIndicators of Radiation Injury in Man (International Atomic EnergyAgency, Vienna, 1971); and Berg, Ed. Genetic Damage in Man Caused byEnvironmental Agents (Academic Press, New York, 1979). A host ofpotentially carcinogenic and teratogenic chemicals are widelydistributed in the environment because of industrial and agriculturalactivity. These chemicals include pesticides, and a range of industrialwastes and by-products, such as halogenated hydrocarbons, vinylchloride, benzene, arsenic, and the like, Kraybill et al., Eds.,Environmental Cancer (Hermisphere Publishing Corporation, New York,1977). Sensitive measures of chromosomal breaks and other abnormalitiescould form the basis of improved dosimetric and risk assessmentmethodologies for evaluating the consequences of exposure to suchoccupational and environmental agents.

Current procedures for genetic screening and biological dosimetryinvolve the analysis of karyotypes. A karyotype is a collection ofindices which characterize the state of an organism's chromosomalcomplement. It includes such things as total chromosome number, copynumber of individual chromosome types (e.g., the number of copies ofchromosome X), and chromosomal morphology, e.g., as measured by length,centromeric index, connectedness, or the like. Chromosomal abnormalitiescan be detected by examination of karyotypes. Karyotypes are determinedby staining an organism's metaphase, or condensed, chromosomes.Metaphase chromosomes are used because, until recently, it has not beenpossible to visualize nonmetaphase, or interphase chromosomes due totheir dispersed condition in the cell nucleus.

The metaphase chromosomes can be stained by a number of cytologicaltechniques to reveal a longitudinal segmentation into entities generallyreferred to as bands. The banding pattern of each chromosome within anorganism is unique, permitting unambiguous chromosome identificationregardless of morphological similarity, Latt, "Optical Studies ofMetaphase Chromosome Organization," Annual Review of Biophysics andBioengineering, Vol. 5, pgs. 1-37 (1976). Adequate karyotyping fordetecting some important chromosomal abnormalities, such astranslocations and inversions requires banding analysis. Unfortunately,such analysis requires cell culturing and preparation of high Qualitymetaphase spreads, which is extremely difficult and time consuming, andalmost impossible for tumor cells.

The sensitivity and resolving power of current methods of karyotyping,are limited by the lack of stains that can readily distinguish differentchromosomes having highly similar staining characteristics because ofsimilarities in such gross features as size, morphology, and/or DNA basecomposition.

In recent years rapid advances nave taken place in the study ofchromosome structure and its relation to genetic content and DNAcomposition. In part, the progress has come in the form of improvedmethods of gene mapping based on the availability of large quantities ofpure DNA and RNA fragments for probes produced by genetic engineeringtechniques, e.g., Kao, "Somatic Cell Genetics and Gene Mappings,"International Review of Cytology, Vol. 85, pgs. 109-146 (1983), andD'Eustachio et al., "Somatic Cell Genetics in Gene Families," Science,Vol. 220, pgs. 9, 19-924 (1983). The probes for gene mapping compriselabeled fragments of single stranded or double stranded DNA or RNA whichare hybridized to complementary sites on chromosomal DNA. The followingreferences are representative of studies utilizing gene probes formapping: Harper et al. "Localization of the Human Insulin Gene to theDistal End of the Short Arm of Chromosome 11," Proc. Natl. Acad. Sci.,Vol. 78, pgs. 4458-4460; Kao et al., "Assignment of the Structural GeneCoding for Albumin to Chromosome 4," Human Genetics, Vol. 62, pgs.337-341 (1982); Willard et al., "Isolation and Characterization of aMajor Tandem Repeat Family from the Human X Chromosome," Nucleic AcidsResearch, Vol. 11, pgs. 2017-2033 (1983); and Falkow et al., U.S. Pat.No. 4,358,535, issued 9 Nov. 1982, entitled "Specific DNA Probes inDiagnostic Microbiology." The hybridization process involvesunravelling, or melting, the double stranded nucleic acids by heating,or other means. This step in the hybridization process is sometimesreferred to as denaturing the nucleic acid. When the mixture of probeand target nucleic acids cool, strands having complementary basesrecombine, or anneal. When a probe anneals with a target nucleic acid,the probe's location on the target can be detected by a label carried bythe probe. When the target nucleic acid remains in its naturalbiological setting, e.g., DNA in chromosomes or cell nuclei (albeitfixed or altered by preparative techniques) the hybridization process isreferred to as in situ hybridization.

Use of hybridization probes has been limited to identifying the locationof genes or known DNA sequences on chromosomes. To this end it has beencrucially important to produce pure, or homogeneous, probes to minimizehybridizations at locations other than at the site of interest,Henderson, "Cytological Hybridization to Mammalian Chromosomes,"International Review of Cytology, Vol. 76, pgs. 1-46 (1982).

Manuelidis et al., in "Chromosomal and Nuclear Distribution of the HindIII 1.9-KB Human DNA Repeat Segment," Chromosoma, Vol. 91, pgs. 28-38(1984), disclose the construction of a single kind of DNA probe fordetecting multiple loci on chromosomes corresponding to members of afamily of repeated DNA sequences.

Wallace et al., in "The Use of Synthetic Oligonucleotides asHybridization Probes. II. Hybridization of Oligonucleotides of MixedSequence to Rabbit Beta-Globin DNA, "Nucleic Acids Research, Vol. 9,pgs. 879-894 (1981), disclose the construction of syntheticOligonucleotide probes having mixed base sequences for detecting asingle locus corresponding to a structural gene. The mixture of basesequences was determined by considering all possible nucleotidesequences which could code for a selected sequence of amino acids in theprotein to which the structural gene corresponded.

Olsen et al., in "Isolation of Unique Sequence Human X ChromosomalDeoxyribonucleic Acid," Biochemistry, Vol. 19, pgs. 241 9-2428 (1980),disclose a method for isolating labeled unique sequence human Xchromosomal DNA by successive hybridizations: first, total genomic humanDNA against itself so that a unique sequence DNA fraction can beisolated; second, the isolated unique sequence human DNA fractionagainst mouse DNA so that homologous mouse/human sequences are removed;and finally, the unique sequence human DNA not homologous to mouseagainst the total genomic DNA of a human/mouse hybrid whose only humanchromosome is chromosome X, so that a fraction of unique sequence Xchromosomal DNA is isolated.

SUMMARY OF THE INVENTION

The invention includes methods and compositions for stainingchromosomes. In particular, chromosome specific staining reagents areprovided which comprise heterogeneous mixtures of labeled nucleic acidfragments having substantial portions of substantially complementarybase sequences to the chromosomal DNA for which specific staining isdesired. The nucleic acid fragments of the heterogenous mixtures includedouble stranded or single stranded RNA or DNA. Heterogeneous inreference to the mixture of labeled nucleic acid fragments means thatthe staining reagents comprise many copies each of fragments havingdifferent base compositions and/or sizes, such that application of thestaining reagent to a chromosome results in a substantially uniformdistribution of fragments hybridized to the chromosomal DNA.

"Substantial proportions" in reference to the basic sequences of nucleicacid fragments that are complementary to chromosomal DNA means that thecomplementarity is extensive enougn so that the fragments form stablehybrids with the chromosomal DNA under standard hybridization conditionsfor the size and complexity of the fragment. In particular, the termcomprehends the situation where the nucleic acid fragments of theheterogeneous mixture possess regions having non-complementary basesequences.

As discussed more fully below, preferably the heterogeneous mixtures aresubstantially free from so-called repetitive sequences, both the tandemvariety and the interspersed variety (see Hood et al., Molecular Biologyof Eucaryotic Cells (Benjamin/Cummings Publishing Company, Menlo Park,Calif., 1975) for an explanation of repetitive sequences). Hood et al.states at pages 47-48 that "[e]ucaryotic sequences can be dividedsomewhat arbitrarily into three general frequency classes, termed highlyrepetitive (also called satellite DNA), middle-repetitive, and unique."Hood et al. indicates at page 49 that "[h]ighly repetitive DNA sequencesare located in regions of centromeric heterochromatin", and at page 50that "[m]iddle-repetitive sequences are interspersed among uniquesequences."

Tandem repeats are so named because they are clustered or contiguous onthe DNA molecule which forms the backbone of a chromosome. Members ofthis class of repeats are also associated with well-defined regions ofthe chromosome, e.g., the centromeric region. Thus, if these repeatsform a sizable fraction of a chromosome, and are removed from theheterogeneous mixture of fragments employed in the invention, perfectuniformity of staining may not be possible. This situation iscomprehended by the use of the term "substantially uniform" in referenceto the heterogeneous mixture of labeled nucleic acid fragments of theinvention.

It is desirable to disable the hybridization capacity of repetitivesequences because copies occur on all the chromosomes of a particularorganism; thus, their presence reduces the chromosome specificity of thestaining reagents of the invention. As discussed more fully belowhybridization capacity can be disabled in several ways, e.g., selectiveremoval or screening of repetitive sequences from chromosome specificDNA, selective blocking of repetitive sequences by pre-reassociationwith complementary fragments, or the like.

Preferably, the staining reagents of the invention are applied tointerphase or metaphase chromosomal DNA by in situ hybridization, andthe chromosomes are identified or classified, i.e., karyotyped, bydetecting the presence of the label on the nucleic acid fragmentscomprising the staining reagent.

The invention includes chromosome staining reagents for the totalgenomic complement of chromosomes, staining reagents specific to singlechromosomes, staining reagents specific to subsets of chromosomes, andstaining reagents specific to subregions within a single chromosome. Theterm "chromosome-specific," is understood to encompass all of theseembodiments of the staining reagents of the invention. The term is alsounderstood to encompass staining reagents made from both normal andabnormal chromosome types.

A preferred method of making the staining reagents of the inventionincludes isolating chromosome-specific DNA, cloning fragments of theisolated chromosome-specific DNA to form a heterogeneous mixture ofnucleic acid fragments, disabling the hybridization capacity of repeatedsequences in the nucleic acid fragments, and labeling the nucleic acidfragments to form a heterogeneous mixture of labeled nucleic acidfragments. As described more fully below, the ordering of the steps forparticular embodiments varies according to the particular means adoptedfor carrying out the steps.

The preferred method of isolating chromosome-specific DNA for cloningincludes isolating specific chromosome types by fluorescence-activatedsorting.

The present invention addresses problems associated with karyotypingchromosomes, especially for diagnostic and dosimetric applications. Inparticular, the invention overcomes problems which arise because of thelack of stains that are sufficiently chromosome-specific by providingreagents comprising heterogeneous mixtures of labeled nucleic acidfragments that-can be hybridized to the DNA of specific Chromosomes,specific subsets of chromosomes, or specific subregions of specificchromosomes. The staining technique of the invention opens up thepossibility of rapid and highly sensitive detection of chromosomalabnormalities in both metaphase and interphase cells using standardclinical and laboratory equipment. It has direct application in geneticscreening, cancer diagnosis, and biological dosimetry.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1a is a binary image of a DAPI (4,6-diamidino-2-phenylindole,stained human metaphase spread.

FIG. 1b is a binary image of fluorescein staining of the same humanmetaphase spread as FIG. 1a.

FIG. 1c is a binary image of the chromosome 21s alone, nonspecificallystained objects having been removed by standard image processingtechniques.

DETAILED DESCRIPTION OF THE INVENTION

The present invention includes compositions for staining individualchromosome types and methods for making and using the compositions. Thecompositions comprise heterogeneous mixtures of labeled nucleic acidfragments. The individual labeled nucleic acid fragments making up theheterogeneous mixture are essentially standard hybridization probes.That is, each chromosome-specific staining composition of the inventioncan be viewed as a large collection of hybridization probes to uniquesequence regions of a specific chromosome. In fact, the preferred methodof making the compositions of the invention entails generating aheterogeneous mixture on a fragment-by-fragment basis by isolating,cloning chromosomal DNA, and selecting from the clones hybridizationprobes to unique sequence regions of a particular chromosome. Theprecise number of distinct labeled nucleic acid fragments, or probes,comprising a heterogeneous mixture is not a critical feature of theinvention. However, in particular applications, a trade-off may nave tobe established between the number of distinct fragments in aheterogeneous mixture and the degree of nonspecific background staining:Where the tendency for nonspecific background staining is nigh, givingrise to low signal to noise ratios, it may De necessary to reduce thenumber of distinct fragments comprising the heterogeneous mixture. Onthe other hand, where nonspecific background staining is low, the numberof distinct fragments may be increased. Preferably, the numbers ofdistinct fragments in a heterogeneous mixture is as high as possible(subject to acceptable signal to noise ratios) so that staining appearscontinuous over the body of the chromosomes.

Another constraint on the number of distinct fragments in theheterogeneous mixture is solubility. Upper bounds exist with respect tothe fragment concentration, i.e., unit length of nucleic acid per unitvolume, that can be maintained in solution. Thus, if fragments of agiven average length are used at a given concentration (fragment pervolume), then the number of different such fragments that can comprisethe heterogeneous mixture is limited.

In one preferred embodiment where the heterogeneous mixture is generatedon a fragment-by-fragment basis, the chromosomal DNA is initially clonedin long sequences, e.g., 35-45 kilobases in cosmids, or like vector.After amplification the inserts are cut into smaller fragments andlabeled for formation to a heterogeneous mixture. In this embodiment,the chromosomal binding sites of the fragments are clustered in thechromosomal regions complementary to the cloned "parent" nucleic acidsequence. Fluorescent signals from such clusters are more readilydetected than signals from an equivalent amount of label dispersed overthe entire chromosome. In this embodiment, the clusters aresubstantially uniformly distributed over the chromosome.

Repetitive sequences, repeated sequences and repeats are usedinterchangeably throughout.

I. Isolation of Chromosome-Specific DNA and Formation of DNA FragmentLibraries.

The first step in the preferred method of making the compositions of theinvention is isolating chromosome-specific DNA. This step includes firstisolating a sufficient quantity of the particular chromosome type towhich the staining composition is directed, then extracting the DNA fromthe isolated chromosomes. Here "sufficient quantity" means sufficientfor carrying out subsequent steps of the method. Preferably, theextracted DNA is used to create a chromosome-specific library of DNAinserts which can be cloned using standard genetic engineeringtechniques. The cloned inserts are then isolated and treated to disablethe hybridization capacity of repeated sequences. In this case,"sufficient quantity" means enough for the particular method used inconstructing the DNA insert library.

Several methods are available for isolating particular chromosome types.For example, a technique for isolating human chromosome types involvesforming hybrid cell lines from human cells and rodent cells,particularly mouse or hamster cells, e.g., see Kao, "Somatic CellGenetics and Gene Mapping," International Review of Cytology. Vol. 85,pgs. 109-146 (1983), for a review. Human chromosomes are preferentiallylost by the hybrid cells so that hybrid cell lines containing a fullcomplement of rodent chromosomes and a single human chromosome can beselected and propagated, e.g., Gusella et al., "Isolation andLocalization of DNA Segments from Specific Human Chromosomes," Proc.Natl Acad Sci., Vol. 77, pgs. 2829-2833 (1980). Chromosome specific DNAcan then be isolated by techniques disclosed by Schmeckpeper et al.,"Partial Purification and Characterization of DNA from Human XChromosome," Proc. Natl Acad Sci., Vol. 76, pgs. 6525-6528 (1979); orOlsen et al., "Isolation of Unique Sequence Human X ChromosomalDeoxyribonucleic Acid," Biochemistry, Vol. 19, pgs. 2419-2428 (1980).Accordingly, these references are incorporated by reference. Briefly,sheared total human DNA is hybridized against itself on hydroxyapatiteunder conditions that allow elution of substantially pure uniquesequence total human DNA from the hydroxyapatite. The unique sequencetotal human DNA is then reassociated and nick translated to add a label(see Maniatis et al., Molecular Cloning A Laboratory Manual, Cold SpringHarbor Laboratory, 1982 , pgs. 109-112, for a description of the nicktranslation technique to add radioactive labels; and Brigato et al.,"Detection of Viral Genomes in Cultured Cells and Paraffin-EmbeddedTissue Sections Using Biotin-Labeled Hybridization Probes," Virology,Vol. 126, pgs. 32-50 (1982) for a description of the nick translationtechnique to add a biotin label). Poly(dA) tails are then synthesized tothe 3' hydroxyl termini of the unique sequence DNA by incubating withterminal transferase under appropriate conditions. The poly(dA) uniquehuman DNA is then hybridized against rodent DNA to remove homologoussequences. The nonhomologous poly(dA) unique human DNA is thenhybridized against the total DNA of the human/rodent hybrid onhydroxyapatite; double stranded DNA is isolated. (This is thechromosome-specific unique sequence human DNA.) The poly(dA) tail isused to separate the labeled unique sequence DNA from the unlabeledunique sequences by binding the poly(dA) tails to oligo(dT) cellulose.

The preferred means for isolating particular chromosome types is bydirect flow sorting of metaphase chromosomes with or without the use ofinterspecific hybrid cell systems. Direct sorting is preferred becausethere is considerable DNA sequence homology between rodent and humanDNA, which necessitates additional hybridization steps (e.g., see Olsenet al., cited above), and the hybrid cell lines tend to be unstable withrespect to retention of the human chromosomes.

For some species, every chromosome can be isolated by currentlyavailable sorting techniques. Most, but not all, human chromosomes arecurrently isolatable by flow sorting, Carrano et al., "Measurement andPurification of Human Chromosomes by Flow Cytometry and Sorting," Proc.Natl. Acad. Sci., Vol. 76, pgs. 1382-1384 (1979). Thus, for isolation ofsome human chromosomes, use of the human/rodent hybrid cell system maybe necessary. Chromosome sorting can be done by commercially availablefluorescence-activated sorting machines, e.g., Becton Dickinson FACS-IIor like instrument.

DNA is extracted from the isolated chromosomes by standard techniques,e.g., Marmur, "A Procedure for the Isolation of Deoxyribonucleic Acidfrom Micro-Organisms," J. Mol. Biol., Vol. 3, pgs. 208-218 (1961); orManiatis et al., Molecular Cloning: A Laboratory Manual (Cold SpringHarbor Laboratory, 1982)pgs. 280-281. These references are incorporatedby reference for their descriptions of DNA isolation techniques.

Generation of insert libraries from the isolated chromosome-specific DNAis carried out using standard genetic engineering techniques, e.g.,Davies et al., "Cloning of a Representative Genomic Library of the HumanX Chromosome After Sorting by Flow Cytometry," Nature, Vol. 293, pgs.374-376 (1981 ); Krumlauf et al., "Construction and Characterization ofGenomic Libraries from Specific Human Chromosomes," Proc. Natl. Acad.Sci., Vol. 79, pgs. 2971-2975 (1982); Lawn et al., "The Isolation andCharacterization of Linked Delta-and-Beta-Globin Genes from a ClonedLibrary of Human DNA." Cell, Vol. 15, pgs. 1157-1174 (1978); andManiatis et al., "Molecular Cloning: A Laboratory Manual," (Cold SpringsHarbor Laboratory, 1982), pgs. 256-308, the cited pages of Maniatis etal. and Lawn et al. being incorporated by reference.

In some cases, it is preferable that the nucleic acid fragments of theheterogeneous mixture consist of single-stranded RNA or DNA. The bindingefficiency of single stranded nucleic acid probes has been found to behigher during in situ hybridization, e.g., Cox et al., "Detection ofmRNAs in Sea Urchin Embryos by In Situ Hybridization Using AsymmetricRNA Probes," Developmental Biology, Vol. 101, pgs. 485-502 (1984).Standard methods are used to generate RNA fragments from isolated DNAfragments. For example, a method developed by Green et al., described inCell, Vol. 32, pgs. 681-694 (1983), is commercially available fromPromega Biotec (Madison, Wis.) under the tradename "Riboprobe." Othertranscription kits suitable for use with the present invention areavailable from United States Biochemical Corporation (Cleveland, Ohio)under the tradename "Genescribe." Single stranded DNA probes can beproduced with the single stranded bacteriophage M13, also available inkit form, e.g. Bethesda Research Laboratories (Gaithersburg, Md.).

II. Disabling the Hybridization Capacity of Repetitive Sequences.

As mentioned above, it is desirable to disable the hybridizationcapacity of repetitive sequences by removal, block, or like means.Repetitive sequences are distributed throughout the genome; most are notchromosome-specific. Consequently, in spite of the fact that the nucleicacid fragments of the invention are derived from isolated chromosomes,the presence of repeats greatly reduces the degree ofchromosome-specificity of the staining reagents of the invention,particularly in genomes containing a significant fraction of repetitivesequences, such as the human genome.

Several techniques are available for disabling the hybridizationcapacity of repetitive sequences. Highly repetitive DNA sequences can beremoved from the extracted chromosome-specific DNA by denaturing andincubating the extracted DNA against itself or against repetitivesequence-enriched total genomic DNA on hydroxyapatite, or likeabsorbent, at low C_(o) t values, followed by fractionation of doublestranded DNA from single stranded DNA.

Hydroxyapatite chromatography is a standard technique for fractionatingDNA on the basis of reassociation conditions such as temperature, saltconcentration, or the like. It is also useful for fractionating DNA onthe basis of reassociation rate at fixed reassociative conditions, orstringencies. Materials for hydroxyapatite chromotography are availablecommercially, e.g., Bio-Rad Laboratories (Richmond, Calif.).

Fractionation based on resistance to S₁ nuclease can also be used toseparate single stranded from double stranded DNA after incubation toparticular C_(o) t values. See Britten et al. "Analysis of Repeating DNASequences by Reassociation," in Methods in Enzymology, Vol. 29, pgs.363-418 (1974), for an explanation of C_(o) t values. Preferably, thisinitial reassociation step is carried out after the extractedchromosome-specific DNA has been broken into pieces and amplified bycloning.

One embodiment of the invention is obtained by labeling the fragments ofthe single stranded fraction eluted from the hydroxyapatite in theinitial reassociation step. The particular C_(o) t values required toseparate middle repetitive and highly repetitive sequences from thechromosome-specific DNA may vary from species to species because ofinter-specific differences in the fraction of the genomic DNA comprisingmiddle and highly repetitive sequences. Most preferably in thisembodiment, the initial reassociation step is to a C_(o) t value in therange of about a few hundred to a few thousand.

In addition to self hybridization or hybridization againstrepetitive-sequence-enriched total genomic DNA, removal of repeats fromfragment mixtures can also be accomplished by hybridization againstimmobilized nigh molecular weight total genomic DNA, following aprocedure described by Brison et al., "General Method for CloningAmplified DNA by Differential Screening with Genomic Probes," Molecularand Cellular Biology, Vol. 2, pgs. 578-587 (1982). Accordingly, thisreference is incorporated by reference. Briefly, the procedure removedrepeats from fragment mixtures in the size range of a few tens of basesto a few hundred bases. Minimally sheared total genomic DNA is bound todiazonium cellulose, or like support. The fragment mixture is thenhydridized against the immobilized DNA to C_(o) t values in the range ofabout 1 to 100. The preferred stringency of the hybridization conditionsmay vary depending on the base composition of the DNA.

The preferred means for disabling hybridization capacity is selectingunique sequence nucleic acid inserts from a chromosome-specific DNAlibrary. For example, following Benton and Davis, "Screening Lambda gtRecombinant Clones by Hybridization to Single Plaques in situ," Science,Vol. 196, pgs. 180-182 (1977), pieces of chromosome-specific DNA areinserted into lambda gt bacteriophage or like vector. The phages areplated on agar plates containing a suitable host bacteria. DNA from theresulting phage plaques is then transferred to a nitrocellulose filterby contacting the filter to the agar plate. The filter is then treatedwith labeled repetitive DNA so that phage plaques containing repetitivesequence DNA can be identified. Those plaques that do not correspond tolabeled spots on the nitrocellulose filter comprise clones which maycontain unique sequence DNA. Clones from these plaques are selected andamplified, radioactively labeled, and hybridized to Southern blots ofgenomic DNA which has been digested with the same enzyme used togenerate the inserted chromosome-specific DNA. Clones carrying uniquesequence inserts are recognized as those that produce a single bandduring Southern analysis.

Another method of disabling the hybridization capacity of repetitive DNAsequences within nucleic acid fragments involves blocking the repetitivesequences by pre-reassociation of fragments with fragments ofrepetitive-sequence-enriched DNA, by pre-reassociation of the target DNAwith fragments of repetitive-sequence-enriched DNA, or pre-reassociationof both the fragments of the heterogeneous mixture and the target DNAwith repetitive-sequence-enriched DNA. The method is generally describedby Sealy et al., "Removal of Repeated Sequences from HybridizationProbes," Nucleic Acid Research, Vol. 13, pgs. 1905-1922 (1985), whichreference is incorporated by reference.

The term pre-reassociation refers to a hybridization step involving thereassociation of unlabeled, repetitive DNA or RNA with the nucleic acidfragments of the heterogeneous mixture just prior to the in situhybridization step, or with the target DNA either just prior to orduring the in situ hybridization step. This treatment results in nucleicacid fragments whose repetitive sequences are blocked by complementaryfragments such that sufficient unique sequence regions remain free forattachment to chromosomal DNA during the in situ hybridization step.

III. Labeling the Nucleic Acid Fragments of the Heterogeneous Mixture.

Several standard techniques are available for labeling single and doublestranded nucleic acid fragments of the heterogeneous mixture. Theyinclude incorporation of radioactive labels, e.g. Harper et al.Chromosoma, Vol. 83, pgs. 431-439 (1981); direct attachment offluorescent labels, e.g. Smith et al., Nucleic Acids Research, Vol. 13,pgs. 2399-2412 (1985), and Connolly et al., Nucleic Acids Research, Vol.13, pgs. 4485-45(92 (1985); and various chemical modifications of thenucleic acid fragments that render them detectable immunochemically orby other affinity reactions, e.g. Tchen et al., "Chemically ModifiedNucleic Acids as Immunodetectable Probes in Hybridization Experiments,"Proc. Natl. Acad. Sci., Vol 81, pgs. 3466-3470 (1984); Richardson etal., "Biotin and Fluorescent Labeling of RNA Using T4 RNA Ligase,"Nucleic Acids Research, Vol. 11, pgs. 6167-6184 (1983); Langer et al.,"Enzymatic Synthesis of Biotin-Labeled Polynucleotides: Novel NucleicAcid Affinity Probes," Proc. Natl. Acad. Sci., Vol. 78, pgs. 6633-6637(1981); Brigati et al., "Detection of Viral Genomes in Cultured Cellsand Paraffin-Embedded Tissue Sections Using Biotin-Labeled HybridizationProbes," Virology, Vol 126, pgs. 32-50 (1 983); Broker et al., "ElectronMicroscopic Visualization of tRNA Genes with Ferritin-Avidin: BiotinLabels," Nucleic Acids Research, Vol. 5, pgs. 363-384 (1978); Bayer etal., "The Use of the Avidin Biotin Complex as a Tool in MolecularBiology," Methods of Biochemical Analysis, Vol. 26, pgs. 1-45 (1980) andKuhlmann, Immunoenzyme Techniques in Cytochemistry (Weinheim, Basel,1984).

All of the labeling techniques disclosed in the above references may bepreferred under particular circumstances. Accordingly, the above-citedreferences are incorporated by reference. Several factors govern thechoice of labeling means, including the effect of the label on the rateof hybridization and binding of the nucleic acid fragments to thechromosomal DNA, the accessibility of the bound probe to labelingmoieties applied after initial hybridization, the nature and intensityof the signal generated by the label, the expense and ease in which thelabel is applied, and the like.

The term labeled nucleic acid fragment as used herein comprehendslabeling means which include chemical modification of nucleic acidfragment by substituting derivatized bases, by forming adducts, or thelike, which after hydridization render the nucleic acid fragmentsdetectable by immunochemical stains or affinity labels, such asbiotin-avidin labeling systems, N-acetoxy-N-2-acetylaminofluorene (AFF)labeling systems, or the like.

For most applications, labeling means which generate fluorescent signalsare preferred.

IV. In Situ Hybridization.

Application of the heterogeneous mixture of the invention to chromosomesis accomplished by standard in situ hybridization techniques. Severalexcellent guides to the technique are available, e.g., Gall an d Pardue,"Nucleic Acid Hybridization in Cytological Preparations," Methods inEnzymology, Vol. 21, pgs. 470-480 (1981 ); Henderson, "CytologicalHybridization to Mammalian Chromosomes," International Review ofCytology, Vol. 76, pgs. 1-46 (1982); and Angerer, et al., "In SituHybridization to Cellular RNAs," in Genetic Engineering: Principles andMethods, Setlow and Hollaender, Eds., Vol. 7, pgs. 43-65 (Plenum Press,New York, 1985). Accordingly, these references are incorporated byreferences.

Three factors influence the staining sensitivity of a heterogeneousmixture of hybridization probes: (1) efficiency of hybridization(fraction of target DNA that can be hybridized by probe), (2) detectionefficiency (i.e., the amount of visible signal that can be obtained froma given amount of hybridization probe), and (3) level of noise producedby nonspecific binding of probe or components of the detection system.

Generally in situ hybridization comprises the following major steps: (1)fixation of tissue or biological structure to be examined, (2)prehybridization treatment of the biological structure to increaseaccessibility of target DNA, and to reduce nonspecific binding, (3)hybridization of the heterogeneous mixture of probe to the DNA in thebiological structure or tissue; (4) posthybridization washes to removeprobe not bound in specific hybrids, and (5) detection of the hybridizedprobes of the heterogeneous mixture. The reagents used in each of thesesteps and their conditions of use vary depending on the particularchromosomes being stained.

The following comments are meant to serve as a guide for applying thegeneral steps listed above. Some experimentation may be required toestablish optimal staining conditions for particular applications.

Fixatives include acid alcohol solutions, acid acetone solutions,Petrunkewitsch's reagent, and various aldehydes such as formaldehyde,paraformaldehyde, glutaraldehyde, or the like. Preferably,ethanol-acetic acid or methanol-acetic acid solutions in about 3:1proportions are used to fix the chromosomes. For cells or chromosomes insuspension, the fixation procedure disclosed by Trask, et al., inScience, Vol. 230, pgs. 1401-1402 (1985), is preferred. Accordingly,Trask, et al., is incorporated by reference. Briefly, K₂ CO₃ anddimethylsuberimidate (DMS) are added (from a 5x concentrated stocksolution, mixed immediately before use) to a suspension containing about5×10⁶ nuclei/ml. Final K₂ CO₃ and DMS concentrations are 20 mM and 3 mM,respectively. After 15 minutes at 25° C., the pH is adjusted from 10.0to 8.0 by the addition of 50 microliters of 100 mM citric acid permilliliter of suspension. Nuclei are washed once by centrifugation (300g, 10 minutes 4° C. in 50 mM kCl, 5 mM Hepes buffer, at pH 9.0, and 10mM MgSO₄).

Preferably, before application of the stain, chromosomes are treatedwith agents to remove proteins. Such agents include enzymes or mildacids. Pronase or proteinase K are the preferred enzymes. Optimizationof deproteinization requires a combination of protease concentration anddigestion time that maximize hybrization, but does not causeunacceptable loss of morphological detail. Optimum conditions varyaccording to chromosome types and method of fixation. Thus, forparticular applications, some experimentation may be required tooptimize protease treatment.

Proteins can al so be removed by mild acid extraction e.g., 0.02-0.2NHCl followed by high temperature (e.g., 70° C.) washes.

In some cases pretreatment with RNase may be desirable to removeresidual RNA from the fixed chromosomes. Such removal can beaccomplished by incubation of chromosomes in 50-100 microgram/milliliterRNase the fixed in 2× SSC (where SSC is a solution of 0.15M NaCL and0.015M sodium citrate) for a period of 1-2 hours at room temperature.

The step of hybridizing the probes of the heterogeneous mixture to thechromosomal DNA involves (1) denaturing the chromosomal DNA so thatprobes can gain access to complementary single stranded regions, and (2)applying the heterogeneous mixture under conditions which allow theprobes to anneal to complementary sites. Methods for denaturationinclude incubation in the presence of high pH, low pH, nigh temperature,or organic solvents such as formamide, tetraalkylammonium halides, orthe like, at various combinations of concentration and temperature. Thepreferred denaturing procedure is incubation for between about 1-10minutes in formamide at a concentration between about 35-95 percent in2×X SSC and at a temperature between about 25°-70° C. Determination ofthe optimal incubation time, concentration, and temperature within theseranges depends on several variables, including the method of fixationand chromosome type.

After the chromosomal DNA is denatured, the denaturing agents areremoved before application of the heterogeneous mixture. Where formamideand neat are the primary denaturing agents, removal is convenientlyaccomplished by plunging the substrate or vessel containing thedenatured chromosomes into an ice bath, and/or by several washes by anice-cold solvent, such as a 70%, 80%, 95% cold ethanol series.

The ambient physiochemical conditions of the chromosomal DNA at the timethe heterogeneous mixture is applied is referred to herein as thehybridization conditions, or annealing conditions. Optimal hybridizationconditions for particular applications depend on several factors,including salt concentration, incubation time of chromosomes in theheterogeneous mixture, and the concentrations, complexities, and lengthsof the probes making up the heterogeneous mixture. Roughly, thehybridization conditions must be sufficiently denaturing to minimizenonspecific binding and hybridizations with excessive numbers of basemismatches. On the other hand, the conditions cannot be so stringent asto reduce hybridizations below detectable levels or to requireexcessively long incubation times. Generally, the hybridizationconditions are much less stringent than the conditions for denaturingthe chromosomal DNA.

The concentrations of probes in the heterogeneous mixture is animportant variable. The concentrations must be high enough so that therespective chromosomal binding sites are saturated in a reasonable time(e.g., within about 18 hours), yet concentrations higher than that justnecessary to achieve saturation should be avoided so that nonspecificbinding is minimized. A preferred concentration range of nucleic acidfragments in the heterogeneous mixture is between about 1-10 nanogramsper kilobase of complexity per milliliter.

The fixed chromosomes can be treated in several ways either during orafter the hybridization step to reduce nonspecific binding of probe DNA.Such treatments include adding a large concentration of nonprobe, or"carrier", DNA to the heterogeneous mixture, using coating solutions,such as Dennardt's solution (Biochem. Biophys. Res. Commun., Vol. 23,pgs. 641-645 (1966), with the heterogeneous mixture, incubating forseveral minutes, e.g., 5-20, in denaturing solvents at a temperature5°-10° C. above the hybridization temperature, and in the case of RNAprobes, mild treatment with single strand RNase (e.g., 5-10 microgramsper millileter RNase) in 2×SSC at room temperature for 1 hour).

V. Making and Using a Staining Reagent Specific to Human Chromosome 21.

A. Isolation of Chromosome 21 and Construction of a Chromosome21-Specific Library

DNA fragments from human chromosome-specific libraries are availablefrom the National Laboratory Gene Library Project through the AmericanType Culture Collection (ATCC), Rockville, Md. Chromosome 21-specificDNA fragments were generated by the procedure described by Fuscoe etal., in "Construction of Fifteen Human Chromosome-Specific DNA Librariesfrom Flow-Purified Chromosomes, Cytogentic Cell Gentics, Vol. 43, pgs.79-86 (1986), which reference is incorporated by reference. Briefly, ahuman diploid fibroblast culture was established from newborn foreskintissue. Chromosomes of the cells were isolated by the MgSO₄ method forvan den Engh et al., Cytometery, Vol. 5, pgs. 108-123 (1984), andstained with the fluorescent dyes. Hoechst 33258 and Chromomycin A3.Chromsome 21 was purified on the Lawrence Livermore National Laboratoryhigh speed sorter, described by Peters et al., Cytometry, Vol. 6, pgs.290-301 (1985).

After sorting, chromosome concentrations were approximately 4×10⁵ /ml.Therefore, prior to DNA extraction, the chromosomes (0.2-1.0×10⁶) wereconcentrated by centrifugation at 40,000×g for 30 minutes at 4° C. Thepellet was then resuspended in 100 microliters of DNA isolation buffer(15 mM NaCl 10 mM EDTA, 10 mM Tris HCl pH 8.0) containing 0.5% SDS and100 micrograms/ml proteinase K. After overnight incubation at 37° C.,the proteins were extracted twice with phenol:chloroform:isoamyl alcohol(25:24:1) and once with chloroform:isoamyl alcohol (24:1). Because ofthe small amounts of DNA, each organic phase was reextracted with asmall amount of 10 mM Tris pH 8.0, 1 mM EDTA (TE). Aqueous layers werecombined and transferred to a Schleicher and Schuell mini-collodionmembrane (#UH020/25) and dialyzed at room temperature against TE for 6-8hours. The purified DNA solution was then digested with 50 units of HindIII (Bethesda Research Laboratories, Inc.) in 50 mM NaCl 10 mM Tris HClpH 7.5, 10 mM MgCl₂, 1 mM dithiothreitol. After 4 hours at 37°, thereaction was stopped by extractions with phenol and chloroform asdescribed above. The acqueous phase was dialyzed against water overnightat 4° C. in a mini-collodion bag and then 2 micrograms of Charon 21Aarms cleaved with Hind III and treated with calf alkaline phosphatase(Boehringer Mannheim) were added. This solution was concentrated undervacuum to a volume of 50-100 microliters and transferred to a 0.5 mlmicrofuge tube where the DNA was precipitated with one-tenth volume 3Msodium acetate pH 5.0 and 2 volumes ethanol. The precipitate wascollected by centrifugation, washed with cold 70% ethanol, and dissolvedin 10 microliters of TE.

After allowing several hours for the DNA to dissolve, 1 microliter of10X ligase buffer (0.5M Tris HCl pH 7.4, 0.1 M MgCl₂, 0.1Mdithiothreitol, 10 mM ATP, 1 mg/ml bovine serum albumin) and 1 unit ofT4 ligase (Bethesda Research Laboratory, Inc.) were added. The ligationreaction was incubated at 10° C. for 16-20 hours and 3 microlitersaliquots were packaged into phage particles using in vitro extractsprepared from E. coli strains BHB 2688 and BHB 2690, described by Hohnin Methods in Enzymology, Vol. 68, pgs. 299-309 (1979) MolecularCloning: A Laboratory Manual, (Cold Spring Harbor Laboratory, New York,1982). Briefly, both extracts were prepared by sonication and combinedat the time of in vivo packaging. These extracts packaged wild-typelambda DNA at an efficiency of 1-5×10⁸ plaque forming units (pfu) permicrogram. The resultant phage were amplified on E. coli LE392 at adensity of approximately 10⁴ pfu/150 mm dish for 8 hours to preventplaques from growing together and to minimize differences in growthrates of different recombinants. The phage were eluted from the agar in10 ml SM buffer (50 mM Tris HCl pH 7.5, 10 mM MgSO₄, 100 mM NaCl 0.01%gelatin) per plate by gentle snaking at 4° C. for 12 hours. The plateswere then rinsed with an additional 4 ml of SM. After pelleting cellulardebris, the phage suspension was stored over chloroform at 4° C.

B. Construction and Use of Chromsome 21-Specific Stain for StainingChromosome 21 of Human Lymphocytes

Clones having unique sequence inserts are isolated by the method ofBenton and Davis, Science, Vol. 196, pgs. 180-182 (1977). Briefly, about1000 recombinant phage are isolated at random from the chromosome21-specific library. These are transferred to nitrocellulose and probedwith nick translated total genomic human DNA.

Of the clones which do not show strong hybridization, approximately 300are picked which contain apparent unique sequence DNA. After theselected clones are amplified, the chromosome 21 insert in each clone is³² p labeled and hybridized to Southern blots of human genomic DNAdigested with the same enzyme used to construct the chromosome 21library, i.e., Hind III. Unique sequence containing clones arerecognized as those that produce a single band during Southern analysis.Roughly, 100 such clones are selected for the heterogeneous mixture. Theunique sequence clones are amplified, the inserts are removed by HindIII digestions, and the inserts are separated from the phage arms by gelelectrophoresis. The probe DNA fragments (i.e., the unique sequenceinserts ) are removed from the gel and biotinylated by nick translation(e.g., by a kit available from Bethesda Research Laboratories). LabeledDNA fragments are separated from the nick translation reaction usingsmall spin columns made in 0.5 ml Eppendorph tubes filled with SephadexG-50 (medium) swollen in 50 mM Tris, 1 mM EDTA, 0.1% SDS, at pH 7.5.Human lymphocyte chromosomes are prepared following Harper et al, Proc.Natl. Acad. Sci., Vol. 78, pgs. 4458-4460 (1981). Metaphase andinterphase cells were washed 3 times in phosphate buffered saline, fixedin methanol-acetic acid (3:1) and dropped onto cleaned microscopeslides. Slides are stored in a nitrogen atmosphere at -20° C.

Slides carrying interphase cells and/or metaphase spreads are removedfrom the nitrogen, heated to 65° C. for 4 hours in air, treated withRNase (100 micrograms/ml for 1 hour at 37° C.), and dehydrated in anethanol series. They are then treated with proteinase K (60 ng/ml at 37°C. for 7.5 minutes) and dehydrated. The proteinase K concentration isadjusted depending on the cell type and enzyme lot so that almost nophase microscopic image of the chromosomes remains on the dry slide. Thehybridization mix consists of (final concentrations) 50 percentformamide, 2X SSC, 10 percent dextran sulfate, 500 micrograms/ml carrierDNA (sonicated herring sperm DNA), and 2.0 microgram/ml biotin-labeledchromsome 21-specific DNA. This mixture is applied to the slides at adensity of 3 microliters/cm under a glass cover slip and sealed withrubber cement. After overnight incubation at 37° C., the slides arewashed at 45° C. (50% formamide-2× SS pH 7, 3 times 3 minutes; followedby 2× SSC pH 7, 5 times 2 minutes) and immersed in BN buffer (0.1M Nabicarbonate, 0.05 percent NP-40, pH 8). The slides are never allowed todry after this point.

The slides are removed from the BN buffer and blocked for 5 minutes atroom temperature with BN buffer containing 5% non-fat dry milk(Carnation) and 0.02% Na Azide (5 microliter/cm² under plasticcoverslips). The coverslips are removed, and excess liquid brieflydrained and fluorescein-avidin DCS (3 microgram/ml in BN buffer with 5%milk and 0.02% NaAzide) is applied (5 microliter/cm²). The samecoverslips are replaced and the slides incubated 20 minutes at 37° C.The slides are then washed 3 times for 2 minutes each in BN buffer at45° C. The intensity of biotin-linked fluorescence is amplified byadding a layer of biotinylated goat anti-avidin antibody (5 microgram/mlin BN buffer with 5% goat serum and 0.02% NaAzide), followed, afterwashing as above, by another layer of fluorescein-avidin DCS.Fluorescein-avidin DCS, goat antiavidin and goat serum are all availablecommercially, e.g., Vector Laboratories (Burlingame, Calif.). Afterwashing in BN, a fluorescence antifade solution, p-phenylenediamine (1.5microliter/cm.sup. 2 of coverslip) is added before observation. It isimportant to keep this layer thin for optimum microscopic imaging. Thisantifade significantly reduced fluorescein fading and allows continuousmicroscopic observation for up to 5 minutes. The DNA counterstains (DAPIor propidium iodide) are included in the antifade at 0.25-0.5microgram/mi.

The red-fluorescing DNA-specific dye propidium iodide (PI) is used toallow simultaneous observation of hybridized probe and total DNA. Thefluorescein and PI are excited at 450-490 nm (Zeiss filter combination487709). Increasing the excitation wavelength to 546 nm (Zeiss filtercombination 487715) allows observation of the PI only. DAPI, a bluefluorescent DNA-specific stain excited in the ultraviolet (Zeiss filtercombination 487701), is used as the counterstain when biotin-labeled andtotal DNA are observed separately. Metaphase chromosome 21s are detectedby randomly located spots of yellow distributed over the body of thechromosome.

VI. Chromosome 21-Specific Staining by Blocking Repetitive Probe andChromosomal DNA

High concentrations of unlabeled human genomic DNA and lambda phage DNAwere used to inhibit the binding of repetitive and vector DNA sequencesto the target chromosomes. Heavy proteinase digestion and subsequentfixation improved access of probes to target DNA.

Human metaphase spreads were prepared on microscope slides with standardtechniques and stored immediately in a nitrogen atmosphere at -20° C.

Slides were removed from the freezer and allowed to warm to roomtemperature in a nitrogen atmosphere before beginning the stainingprocedure. The warmed slides were first treated with 0.6 microgram/mlproteinase K in P buffer (20 mM Tris, 2 mM CaCl₂ at pH 7.5) for 7.5minutes, and washed once in P buffer. The amount of proteinase K usedneeds to be adjusted for the particular enzyme lot and cell type. Nextthe slides were washed once in paraformaldehyde buffer (phosphatebuffered saline (PBS) plus 50 mM MgCl₂, at pH 7.5), immersed for 10minutes in 4% paraformaldehyde in paraformaldehyde buffer, and washedonce in 2× SSC (0.3M NaCl, 0.03 M sodium citrate at pH 7). DNA on theslides was denatured by immersing in 70% formamide and 2× SSC at 70° C.for 2 minutes. After denaturing the slides were stored in 2× SSC. Ahybridization mix was prepared which consisted of 50% formamide, 10%dextran sulfate, 1% Tween 20, 2× SSC, 0.5 mg/ml human genomic DNA, 0.03mg/ml lambda DNA, and 3 microgram/ml biotin labeled probe DNA. The probeDNA consisted of the highest density fraction of phage from thechromosome 21 Hind III fragment library (ATCC accession number 57713),as determined by a cesium chloride gradient. (Both insert and phage DNAof the probe were labeled by nick translation.) The average insert size(amount of chromosome 21 DNA), as determined by gel electrophoresis isabout 5 kilobases. No attempt was made to remove repetitive sequencesfrom the inserts or to isolate the inserts from the lambda phage vector.The hybridization mix was denatured by heating to 70° C. for 5 minutesfollowed by incubation at 37° C. for 1 hour. The incubation allows thehuman genomic DNA and unlabeled lambda DNA in the hybridization mix toblock the human repetitive sequences and vector sequences in the probe.

The slide containing the human metaphase spread was removed from the 2×SSC and blotted dry with lens paper. The hybridization mix wasimmediately applied to the slide, a glass cover slip was placed on theslide with rubber cement, and the slide was incubated overnight at 37°C. Afterwards preparation of the slides proceeded as described inSection V (wherein chromosome 21 DNA was stained with fluorescein andtotal chromosomal DNA counterstained with DAPI). FIGS. 1a-c illustratethe results. FIG. 1a is a DAPI image of the human metaphase spreadobtained with a computerized image analysis system. It is a binary imageshowing everything above threshold as white, and the rest as black. Theprimary data was recorded as a gray level image with 256 intensitylevels. (Small arrows indicate the locations of the chromosome 21S.)FIG. 1b is a fluorescein image of the same spread as in FIG. 1a, againin binary form. (Again, small arrows indicate the locations of thechromosome 21s.) FIG. 1c illustrates the positions of the chromosome 21swith other less densely stained objects removed standard by imageprocessing techniques.

The descriptions of the foregoing embodiments of the invention nave beenpresented for purpose of illustration and description. They are notintended to be exhaustive or to limit the invention to the precise formdisclosed, and obviously many modifications and variations are possiblein light of the above teaching. The embodiments were chosen anddescribed in order to best explain the principles of the invention andits practical application to thereby enable others skilled in the art tobest utilize the invention in various embodiments and with variousmodifications as are suited to the particular use contemplated. It isintended that the scope of the invention be defined by the claimsappended hereto.

We claim:
 1. A method of staining target chromosomal DNA comprising:(a)providing 1) labeled nucleic acid that comprises fragments which aresubstantially complementary to nucleic acid segments within thechromosomal DNA for which detection is desired, and 2) blocking nucleicacid that comprises fragments which are substantially complementary torepetitive segments in the labeled nucleic acid; and (b) employing saidlabeled nucleic acid, blocking nucleic acid, and chromosomal DNA in insitu hybridization so that labeled repetitive segments are substantiallyblocked from binding to the chromosomal DNA, while hybridization ofunique segments within the labeled nucleic acid to the chromosomal DNAis allowed, wherein blocking of the labeled repetitive segments issufficient to permit detection of hybridized labeled nucleic acidcontaining unique segments, and wherein the chromosomal DNA is presentin a morphologically identifiable chromosome or cell nucleus during thein situ hybridization.
 2. The method of claim 1 wherein the blockingnucleic acid is hybridized with the labeled nucleic acid prior tohybridization of the labeled nucleic acid with the chromosomal DNA. 3.The method of claim 1 wherein the blocking nucleic acid is hybridizedwith the chromosomal DNA prior to hybridization of the labeled nucleicacid with the chromosomal DNA.
 4. The method of claim 1 wherein theblocking nucleic acid is hybridized with the labeled nucleic acid andseparately with the chromosomal DNA prior to hybridization of thelabeled nucleic acid with the chromosomal DNA.
 5. The method of claim 1wherein the labeled nucleic acid, the blocking nucleic acid, and thechromosomal DNA are hybridized together during in situ hybridizationstep.
 6. The method of claim 1 wherein the labeled nucleic acidcomprises fragments which are designed to allow detection of extra ormissing chromosomes, extra or missing portions of a chromosome, orchromosomal rearrangements.
 7. The method of claim 6 wherein thechromosomal rearrangement is a translocation or an inversion.
 8. Themethod of claim 6 wherein the labeled nucleic acid is designed to allowdetection of a chromosomal rearrangement consistent with chronicmyelogenous leukemia.
 9. The method of claim 6 wherein the labelednucleic acid is designed to allow detection of aneuploidy.
 10. Themethod of claim 6 wherein the extra chromosome is chromosome
 21. 11. Themethod of claim 1 wherein the labeled nucleic acid comprises fragmentscomplementary to the total genomic complement of chromosomes, fragmentscomplementary to a single chromosome, fragments complementary to asubset of chromosomes, or fragments complementary to a subregion of asingle chromosome.
 12. The method of claim 11 wherein the fragments areselected from the nucleic acid of normal human chromosomes 1 through 22,X, and Y.
 13. The method of claim 1 wherein the chromosomal DNA ismetaphase or interphase chromosomal DNA.
 14. The method of claim 1wherein the repetitive segments comprise segments which aresubstantially complementary to highly repetitive segments andmiddle-repetitive segments.
 15. The method of claim 14 wherein therepetitive segments are substantially complementary to satelliterepetitive segments.
 16. The method of claim 14 wherein the repetitivesegments are substantially complementary to tandem repetitive segmentsor repetitive segments located in regions of centromericheterochromatin.
 17. The method of claim 14 wherein the repetitivesegments are substantially complementary to interspersed repetitivesegments.